Diabetes Mellitus:- Part 5 – Glucose in Urine (Glycosuria), Urine Sugar, Benedict’s solution

Sample

1. The test sample is urine.
2. The best sample after 2 to 3 hours of the meal.

Indications

1. To diagnose diabetes mellitus.
2. To monitor the diabetes mellitus.
3. To evaluate the effectiveness of the therapy.
4. To diagnose gestational diabetes.
5. It is part of a routine urine examination.

Precautions

1. Can see false-positive tests when any substance present in the urine can reduce the copper in the clinitest strips.
2. Can see the false positive tests in the presence of other sugars like galactose, fructose, and lactose.
3. Drugs giving false positive tests are acetylsalicylic acid, Ascorbic acid, cephalothin, chloral hydrate, streptomycin, sulphonamides, and aminosalicylic acid.
   1. Contamination from the oxidizing agents or bleach.
   2. Improper storage of the urine strips.
4. Drugs giving false-negative results are levodopa, phenazopyridine, and ascorbic acid with clinitest strips.
   1. Multistix detects ≥50 mg/dL, if the amount is less then it will be negative.
   2. Sodium fluoride cause enzyme inhibition.
   3. Refrigerated specimens give decreased enzyme activity.
5. There are drugs that give increased glucose in the urine like diuretics (thiazide), estrogens, isoniazid, lithium, nalidixic acid, nicotinic acid, chloramphenicol, chloral hydrate, cephalosporin, and aminosalicylic acid.

Pathophysiology

1. Examination of urine for glucose is rapid, noninvasive, and inexpensive for screening the urine.
   1. A large number of urine samples can be tested.
2. Glucose is filtered by the glomeruli and reabsorbed by proximal convoluted tubules.
3. When glucose exceeds the renal threshold level(>180 mg/dL) of tubules then, glucose appears in the urine is called glycosuria.
4. Tubular absorption is an active process to maintain the body’s glucose level.
   1. Renal threshold for glucose = 160 to 180 mg/dL.
5. After the renal threshold values glucose appears in the urine.
6. This is not sensitive nor specific for the control of diabetes because we don’t know about the glucose level below 180 mg/dL.
   

   Renal glycosuria mechanism

   List of reducing substances in the urine are:

   1. Lactose.
   2. Fructose.
   3. Galactose.
   4. Maltose.
   5. Arabinose.
   6. Xylose.
   7. Ribose.
   8. Other substances are:
      1. Uric acid.
      2. Creatinine.
      3. Cysteine.
      4. Ketone bodies.
      5. Oxalic acid.
      6. Glucuronic acid.
      7. Hippuric acid.
      8. Homogentisic acid.
      9. drugs are:
         1. Ascorbic acid.
         2. Isoniazid.
         3. Salicylates.
         4. Formaldehyde.

Normal

Source 2

1. Normally sugar (glucose ) is absent in the urine.
   1. Random specimen = negative
2. 24 hours specimen = < 0.5 g/day (<2.78 mmol/day).
3. Glucose appears in the urine when a blood glucose level of 180 mg/dL or more (cross the renal threshold).
4. Its concentration in the urine correlates with the blood glucose level.

Procedure for glucose in the urine:

Benedict’s solution reagents:

1. It consists of:
   1. CuSO4 (cupric sulphate) = 17.3 grams
   2.  Sodium citrate (Na₃C₆H₅O₇-2H₂O) = 173 grams
   3. Sodium carbonate (Na₂CO₃) = 100 grams
   4. Distle water = 1000 mL
2. Procedure to prepare the solution:
   1. Dissolve cupric sulfate in hot water of 100 mL.
   2. Now dissolve separately sodium citrate and sodium carbonate with heating water of 800 mL. Let it cool.
   3. Mix solutions 1 and 2 and makeup to 1000 ml of water.
   4. Benedict’s reagent is ready and is stable.
3. Benedict’s qualitative method is very common.
   1. It contains cupric ions complexed to citrate in an alkaline medium.
   2. Reducing substances convert cupric to cuprous ion.
      1. It forms yellow cuprous hydroxide or red cuprous oxide.
         

         Benedict’s reaction

   3. Benedict’s solution method:
   4. This can be done on the solution of benedicts as well.
      1. Take 2.5 mL Benedict’s solution.
      2. Add 0.2 ml of the urine.
      3. Place the tubes in heat-block or heat directly to bring to 100 °C.
      4. Examine each tube color and the precipitate.
      5. Different colors develop according to the quantity of sugar in the urine.
      6. Greenish brown when there is a large quantity >2 g/dL.
   5. Another way to do Benedict’s reaction:
      1. Benedict’s solution 5 mL.
      2. Urine  0.4 mL (8 drops) .
      3. Mix and keep in a boiling water bath for 3 minutes.
   6. Tablet method:
   7. 5 drops of the urine are mixed with 10 drops of water.
      1. Then add the tablet as known clinitest.
   8. Procedure for Benedict reaction with tablet (Clinitest) shown in the following diagram.
      1. Also, run the negative control as well.
         

         Benedict’s reaction to reducing substances

Reporting the result:

1. It can be reported as plus + signs, from 1+ to 4+.
2. It can report the percentage of 1 to 2 %. This reporting is more accurate.

Color of the urine after Benedict’s reaction	Reporting method	The concentration of glucose
Blue, clear or cloudy (Benedict’s solution color)	0	NIL
Green and no precipitate (may see precipitate)	1+	Traces
Brown and cloudy	2+	Around 0.5 g%
Orange and cloudy	3+	Around 1.o g%
Red and cloudy	4+	Around 2.0 g% or more

2. A semiquantitative method where there are different strips is available like Clinistix, Diastix, and Chemstrip.
   1. In all the above strips, glucose-specific enzyme glucose oxidase is used.
   2. This is more specific for glucose than Benedict’s method.
      1. This test is positive when glucose concentration is 100 mg/dL or more.
   3. The sensitivity of the commercially available Discs:
      1. Multistix = 75 to 125 mg/dL
      2. Diastix  =  75 to 125 mg/dL
      3. Chemstrip = 40 mg/dL in 90% of the specimen.
   4. The drawbacks of the strips:
      1. Urine strips detect mainly glucose, so there are chances for false-negative results due to interfering chemicals in the urine.
      2. False-positive results are seen in:
         1. If the container is contaminated by the detergents.
         2. In very dilute urine, traces may be seen due to sensitivity at low specific gravity.
         3. If the strips are exposed to air due to improper storage.
      3. False-negative results are seen in:
         1. It is seen in the intake of vitamin C (ascorbic acid), ad tetracyclines.
         2. In case if there are high ketone bodies (≥40 mg/dL)  and low glucose level (75 to 125 mg/dL).
         3. Sodium fluoride inhibits the enzyme reaction, so it should not be used in the urine as a preservative.
         4. If urine is refrigerated then it will give false-negative results because of decreased enzyme activity, so bring the urine at room temperature
3. The quantitative method uses hexokinase or glucose dehydrogenase procedure.

Increased glucose in urine seen in:

1. Diabetes mellitus.
2. Renal glycosuria.
3. Hereditary defects in the metabolism of other reducing substances like galactose, pentose, and fructose.
4. Pregnancy.
5. Liver diseases.
6. Pancreatic diseases.
7. Thyrotoxicosis.
8. Cushing’s syndrome.
9. Acromegaly.
10. Brain injuries.
11. Shock.
12. Fanconi’s syndrome (Tubular defect).
13. Advanced renal tubular diseases.
14. Nephrotoxic chemicals like carbon monoxide, lead, and mercury.

The false-negative result is seen in:

1. Mostly seen due to drugs.
   1. Ascorbic acid.
   2. Levodopa.
   3. Phenothiazine.
      Comparison of Benedict reaction and Oxidase method:

      Characteristics	Benedict reaction (CuSO4)	Glucose oxidase
      Minimum level detected	Glucose 50 to 250 mg/dL	Glucose 50 mg/dL
      Other sugars detected	Galactose Lactose Fructose Pentose Maltose	Specific for glucose
      False-negative		Ascorbic acid (Vitamin C) A large number of salicylates Homogentisic acid
      False-positive	Vitamin C (Ascorbic acid) Salicylates Many antibiotics Levodopa X-ray contrast media Homogentisic acid

• Please see more details on Fasting Blood Glucose

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